The present invention is directed to a bacterial delivery system for delivering alphavirus replicon DNA into an animal or animal cells with the replicon encoding one or more heterologous genes to be expressed in the animal or the animal cells. The bacteria are invasive bacteria or attenuated invasive bacteria engineered to contain a DNA vector that encodes the alphavirus replicon in a eukaryotic expression cassette. Upon bacterial infection, primary transcription of the DNA vector is driven by the eukaryotic expression vector and produces an alphavirus replicon RNA which is transported to the cytoplasm. That RNA is transcribed, and translated if the gene encodes a protein, to express the heterologous gene encoded in the alphavirus replicon. The heterologous gene may encode an antigen, a therapeutic agent, an immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody or any other molecule desired for delivery to an animal or animal cell. In a preferred embodiment, the heterologous gene encodes one or more antigens useful as a vaccine for HIV. In addition to the bacterial delivery system, the invention provides methods of introducing and expressing the heterologous gene(s) in animal or animal cells, methods of stimulating or inducing an immune response and vaccines therefor.
There are many applications for delivering DNA to animals or animal cells including for gene therapy of acquired or inherited diseases or conditions, for DNA-based vaccination, for understanding genetic structure and for studying the molecular mechanisms underlying gene expression.
Successful delivery of DNA to animal tissue has been achieved by cationic liposomes [Watanabe et al., Mol. Reprod. Dev. 38:268-274 (1994)], direct injection of naked DNA into animal muscle tissue [Robinson et al., Vacc. 11:957-960 (1993); Hoffman et al., Vacc. 12:1529-1533; (1994); Xiang et al., Virol. 199:132-140 (1994); Webster et al., Vacc. 12:1495-1498 (1994); Davis et al., Vacc. 12:1503-1509 (1994); and Davis et al., Hum. Molec. Gen. 2:1847-1851 (1993)], and embryos [Naito et al., Mol. Reprod. Dev. 39:153-161 (1994); and Burdon et al., Mol. Reprod. Dev. 33:436-442 (1992)], or intradermal injection of DNA using xe2x80x9cgene gunxe2x80x9d technology [Johnston et al., Meth. Cell Biol. 43:353-365 (1994)]. A limitation of these techniques is that they only efficiently deliver DNA to parenteral sites. At present, effective delivery of eukaryotic expression cassettes to mucosal tissue has been met with limited success. This is presumably due to poor access to these sites, toxicity of the delivery vehicles or instability of the delivery vehicles when delivered orally.
For DNA-based vaccination, delivery by injection of naked plasmid DNA has shown potential in mouse models for inducing both humoral and cellular immune responses. However, in larger animals, using DNA delivery for vaccination has been hampered by requiring large amounts of DNA or inducing persistent expression of an antigen with the potential for developing tolerance to the antigen. Berglund reported a strategy for inducing or enhancing an immune response by injecting mice with plasmid DNA containing an alphavirus DNA expression vector having a recombinant Semliki Forest Virus (SFV) replicon in a eukaryotic expression cassette [Berglund et al., Nature Biotechnol. 16:562-565 (1998)]. The eukaryotic expression cassette controlled expression of the primary nuclear transcription of the SFV replicon. This SFV replicon transcript, encoding the heterologous antigen, was transported to the cytoplasm and amplified by the self-encoded SFV replicase complex. The amplified RNA replicon lead to high level production of an mRNA encoding the heterologous antigen. Similar results were described by Polo and his group [Polo et al., Nature Biotechnol. 16:517-518 (1998); Hariharan et al., J. Virol. 72:950-958 (1998)]. Both groups found strong immune responses could be induced using small amounts of input plasmid DNA. Although this method allows greater expression from the input DNA vector, the method still has the disadvantages associated with parenteral delivery.
Alternatively, a method to deliver DNA to animals that overcomes the disadvantages of conventional delivery methods is by administering attenuated, invasive bacteria containing a bacterial DNA vector having a eukaryotic expression cassette encoding the gene to be expressed. For example, U.S. Pat. No. 5,877,159 to Powell et al., describes live bacteria that can invade animal cells without establishing a productive infection or causing disease to thereby introduce a eukaryotic expression cassette encoding an antigen capable of being expressed by the animal cells. While this method allows delivery of the DNA vaccine to mucosal surfaces and is easy to administer, a concern for vaccine delivery in developing countries, it does not have the advantage of providing amplifiable mRNA encoding the gene of interest.
Accordingly, the present invention combines use of live, attenuated invasive bacteria with a eukaryotic expression cassette encoding an alphavirus replicon to provide improved bacterial delivery systems to deliver one or more heterologous genes to an animal. Such systems have the advantages of both bacterial delivery systems and alphavirus replicon vectors and are efficacious, cost effective and safe. The bacterial delivery systems of the invention are particularly useful for delivering DNA for gene therapy and vaccinations.
All cited references and patents are incorporated by reference herein in their entirety.
In accordance with the invention, one embodiment is directed to a bacterial delivery system which comprises a live, invasive bacteria containing a DNA comprising a eukaryotic expression cassette operably linked to an alphavirus replicon DNA capable of amplification as RNA in animal cells, wherein the alphavirus replicon DNA comprises at least one nucleic acid control sequence operably linked to a heterologous nucleic acid sequence to control expression of the heterologous nucleic acid sequence. The live invasive bacteria of the invention are attenuated as needed so long as the bacteria delivered into animals either does not make the animal sick or, at most, causes a self-limiting infection that is clinically innocuous in the animal.
The heterologous nucleic acid sequence can encode an antigen, an antigenic fragment of a protein, a therapeutic agent, an immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a protein, a peptide , an antibody, an antigen binding fragment of an antibody, or any other molecule encodable by DNA and desired for delivery to an animal or animal cell. The heterologous nucleic acid sequence can be obtained from a virus selected from the group consisting of influenza virus, respiratory syncytial virus, HPV, HBV, HCV, HIV, HSV, FeLV, FIV, HTLV-I, HTLV-II, and CMV. The viral sequence can encode one or more viral genes or antigenic fragments thereof. The heterologous nucleotide sequence can encode a cytokine, an interleukin, erythropoietin or other immunostimulatory or immunoregulatory protein.
Another embodiment of this invention is directed to a method for introducing and expressing a gene in an animal by infecting the animal with live, invasive bacteria, preferably attenuated invasive bacteria. These bacteria contain a DNA comprising a eukaryotic expression cassette having an alphavirus replicon DNA capable of amplification as RNA in cells of the animal. Further, the alphavirus replicon DNA directs expression of the one or more heterologous nucleic acid sequences which encode the gene product to be expressed in the animal. The method is applicable to deliver genes encoding an antigen, an antigenic protein fragment, a therapeutic agent, an immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody or any other molecule encodable by DNA and desired for delivery to an animal or an animal cell.
Another aspect of the invention provides a method for inducing an immune response in an animal which by infecting the animal with live, invasive bacteria containing a DNA comprising a eukaryotic expression cassette operably linked to an alphavirus replicon DNA capable of amplification as RNA in animal cells, wherein the alphavirus replicon DNA encodes at least one antigen or antigenic fragment of a protein. The antigen or antigenic fragment are expressed at a level sufficient to stimulate an immune response to the antigen or antigenic fragment. In a preferred embodiment, the antigen is derived from a virus, and more preferably from an HIV virus. This method provides a means to immunize animals, preferably humans, against HIV infection. In a preferred embodiment the, the alphavirus replicon DNA encodes at least one antigen or antigenic fragment from each of the HIV genes env, gag, pol, nef, tat and rev.
Yet another aspect of the invention relates to a method for introducing and expressing a gene in animal cells by (a) infecting animal cells with live, invasive bacteria containing one or more DNA molecules, wherein the DNA molecules comprise a eukaryotic expression cassette operably linked to an alphavirus replicon DNA capable of amplification as RNA in the animal cells and the alphavirus replicon in turn comprises at least one nucleic acid control sequence operably linked to a heterologous nucleic acid sequence to control expression thereof; and (b) culturing those cells under conditions sufficient to express a gene product encoded by said heterologous nucleic acid sequence.